Inhibition of ABCB1 and ABCG2 at the mouse blood-brain barrier with

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Inhibition of ABCB1 and ABCG2 at the mouse bloodbrain barrier with marketed drugs to improve brain delivery of the model ABCB1/ABCG2 substrate [11C]erlotinib Alexander Traxl, Severin Mairinger, Thomas Filip, Michael Sauberer, Johann Stanek, Stefan Poschner, Walter Jäger, Viktoria Zoufal, Gaia Novarino, Nicolas Tournier, Martin Bauer, Thomas Wanek, and Oliver Langer Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01217 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on February 9, 2019

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Molecular Pharmaceutics

Inhibition of ABCB1 and ABCG2 at the mouse blood-brain barrier with marketed drugs to improve brain delivery of the model ABCB1/ABCG2 substrate [11C]erlotinib Alexander Traxl,1 Severin Mairinger,1 Thomas Filip,1 Michael Sauberer,1 Johann Stanek,1 Stefan Poschner,2 Walter Jäger,2 Viktoria Zoufal,1 Gaia Novarino,3 Nicolas Tournier,4 Martin Bauer,5 Thomas Wanek,1 Oliver Langer1,5,6,*

1

Center for Health & Bioresources, AIT Austrian Institute of Technology GmbH,

Seibersdorf, Austria 2

Department of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria

3

Institute of Science and Technology (IST) Austria, Klosterneuburg, Austria

4

UMR 1023 IMIV, Service Hospitalier Frédéric Joliot, CEA, Inserm, Univ. Paris Sud,

CNRS, Université Paris-Saclay, Orsay, France 5 6

Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria Department of Biomedical Imaging und Image-guided Therapy, Division of Nuclear

Medicine, Medical University of Vienna, Vienna, Austria

Corresponding author: Oliver Langer, Biomedical Systems, Center for Health & Bioresources, AIT Austrian Institute of Technology GmbH, 2444 Seibersdorf, Austria, Tel.: +43(0) 50550 3496, E-mail: [email protected]

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Abstract P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are two efflux transporters at the blood-brain barrier (BBB), which effectively restrict brain distribution of diverse drugs, such as tyrosine kinase inhibitors. There is a crucial need for pharmacological ABCB1 and ABCG2 inhibition protocols for a more effective treatment of brain diseases. In the present study, seven marketed drugs (osimertinib, erlotinib, nilotinib, imatinib, lapatinib, pazopanib and cyclosporine A) and one non-marketed drug (tariquidar), with known in vitro ABCB1/ABCG2 inhibitory properties, were screened for their inhibitory potency at the BBB in vivo. Positron emission tomography (PET) using the model ABCB1/ABCG2 substrate [11C]erlotinib was performed in mice. Tested inhibitors were administered as i.v. bolus injections at 30 min before start of the PET scan followed by a continuous i.v. infusion for the duration of the PET scan. Five of the tested drugs increased total distribution volume of [11C]erlotinib in the brain (VT,brain) compared to vehicle-treated animals (tariquidar: +69%, erlotinib: +19% and +23% for the 21.5 mg/kg and the 43 mg/kg dose, respectively, imatinib: +22%, lapatinib: +25% and cyclosporine A: +49%). For all drugs, increases in [11C]erlotinib brain distribution were lower than in Abcb1a/b(-/-)Abcg2(-/-) mice (+149%), which suggested that only partial ABCB1/ABCG2 inhibition was reached at the mouse BBB. The plasma concentrations of the tested drugs at the time of the PET scan were higher than clinically achievable plasma concentrations. Some of the tested drugs led to significant increases in blood radioactivity concentrations measured at the end of the PET scan (erlotinib: +103% and +113% for the 21.5 mg/kg and the 43 mg/kg dose, respectively, imatinib: +125% and cyclosporine A: +101%), which was most likely caused by decreased hepatobiliary excretion of radioactivity. Taken together, our data suggest that some marketed tyrosine kinase inhibitors may be repurposed to inhibit ABCB1 and ABCG2 at the BBB. From a clinical perspective, moderate increases in brain delivery despite the administration of high i.v. doses

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as well as peripheral drug-drug interactions due to transporter inhibition in clearance organs question the translatability of this concept.

Keywords: blood-brain barrier, P-glycoprotein, breast cancer resistance protein, positron emission tomography, [11C]erlotinib, tyrosine kinase inhibitors

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Introduction The brain is a pharmacological sanctuary tissue, as it is sealed from surrounding blood by the blood-brain barrier (BBB).1 The BBB is formed by endothelial cells of the capillary wall, astrocyte end-feet surrounding the capillary, and pericytes embedded in the capillary basement membrane. The brain capillary endothelial cells are connected by tight junctions, which greatly limit paracellular diffusion of solutes from blood into brain.1 Moreover, the ATP-binding cassette (ABC) transporters P-glycoprotein (humans: ABCB1, rodents: ABCB1A/B) and breast cancer resistance protein (ABCG2) impede the transcellular diffusion of a wide range of chemically diverse solutes, including many different drug molecules, across the BBB. ABCB1 and ABCG2 were shown to work together at the BBB in restricting brain entry of dual substrate drugs, which only gain brain access when both transporters are absent or inhibited.2,

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Efflux transport by ABCB1 and ABCG2 affects an

extensive list of small drug molecules and can be considered a major problem in central nervous system (CNS) drug discovery. Certain drug classes, such as molecularly targeted anticancer drugs, appear to be particularly susceptible to ABCB1/ABCG2 transport resulting in poor brain penetration and lack of efficacy for treatment of primary and secondary brain tumors.4-6 The concept of inhibiting ABCB1 to reverse multidrug resistance and improve tumor delivery of anticancer drugs has been intensely pursued in the past and yielded potent experimental ABCB1 inhibitors.7 Clinical trials with these ABCB1 inhibitors in cancer patients have had limited success so that further clinical development of these compounds was stopped.4 Some of these compounds have been repurposed to inhibit ABCB1 at the BBB to enhance brain delivery of CNS-targeted drugs. Preclinical studies have shown that experimental inhibitors, such as valspodar, elacridar and tariquidar, can increase brain penetration of diverse drugs, which in some cases was shown to lead to increased therapeutic

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efficacy.8-12 Nevertheless, none of these inhibitors has received market authorization which severely hampers their clinical applicability. Moreover, to enhance brain penetration of dual ABCB1/ABCG2 substrate drugs both ABCB1 and ABCG2 need to be inhibited,13 which cannot be achieved with most currently available experimental inhibitors. One exception is elacridar, which has proven effective in preclinical species to inhibit ABCB1 and ABCG2 at the BBB.5, 14-17 For instance, we have shown that elacridar can increase in wild-type mice the brain uptake of the radiolabeled epidermal growth factor receptor (EGFR) targeted tyrosine kinase inhibitor (TKI) [11C]erlotinib, which is a dual substrate of ABCB1 and ABCG2, to similar levels as in Abcb1a/b(-/-)Abcg2(-/-) mice, suggesting that elacridar can completely block ABCB1 and ABCG2 at the murine BBB.14 However, as elacridar has a very low oral bioavailability, an intravenous (i.v.) formulation would be required to achieve high enough plasma concentrations for inhibition of ABCB1 and ABCG2 at the human BBB, which is not currently available.15 The development of an i.v. formulation of elacridar is hampered by its very low aqueous solubility.18 There is consequently an unmet clinical need for a dual ABCB1/ABCG2 inhibitor, which is ideally a marketed drug, which can be co-administered with ABCB1/ABCG2 substrate drugs to enhance their brain delivery. Numerous studies have shown that several marketed TKIs are potent ABCB1/ABCG2 inhibitors.19-23 However, most work done so far has been performed in vitro in cell culture and little data is available to prove that TKIs can achieve ABCB1/ABCG2 inhibition in vivo at the BBB.24-26 Erlotinib is a substrate of ABCB1 and ABCG2 at low concentrations,2, inhibits both transporters at higher concentrations.19,

28

27

which

We have recently shown that

supratherapeutic dose administration of erlotinib partially saturates ABCB1/ABCG2 activity at the BBB of mice, non-human primates and humans leading to a non-linear increase in erlotinib brain distribution measured with positron emission tomography (PET) and [11C]erlotinib.14, 15, 29

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In the present study we used PET imaging with [11C]erlotinib as a model ABCB1/ABCG2 substrate in FVB mice to screen marketed drugs with known ABCB1/ABCG2 inhibitory properties for their ability to enhance brain uptake of [11C]erlotinib in vivo. Based on our previous results,14,

15, 25

we included erlotinib as a

benchmark inhibitor. We took advantage of the ability of small-animal PET to perform whole-body PET imaging in mice to also assess the effects of the tested drugs on hepatobiliary and renal excretion of radioactivity.

Experimental Section Chemicals and drugs Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (Schnelldorf, Germany) or Merck (Darmstadt, Germany). Osimertinib was purchased from Axon Medchem (Groningen, The Netherlands), tariquidar dimesylate from Haoyuan Chemexpress Co., Ltd (Shanghai, PRC), erlotinib hydrochloride from Apollo Scientific (Bredbury, UK) and nilotinib monohydrochloride monohydrate, lapatinib ditosylate and pazopanib from Toronto Research Chemicals (North York, ON, Canada). Captisol® was obtained from Cydex Pharmaceuticals (San Diego, CA, USA). For i.v. administration, osimertinib was dissolved in sterile water containing 1% (v/v) 1 M hydrochloric acid (= vehicle 1, pH: 2.5-3.0). Tariquidar dimesylate was dissolved in 2.5% (w/v) aqueous dextrose solution. Erlotinib hydrochloride and nilotinib monohydrochloride monohydrate were dissolved in 2.5% (w/v) aqueous dextrose solution containing 3% (w/v) Captisol® and 1% (v/v) 1 M hydrochloric acid (= vehicle 2, pH: 2.5-3.0). Imatinib mesylate, lapatinib ditosylate, pazopanib and cyclosporine A were dissolved in a mixture of physiological saline (0.9%, w/v)/ethanol/Kolliphor® EL (81/10/9, v/v/w) (= vehicle 3, pH: 6.0-6.5). In the case of pazopanib, vehicle 3 was acidified with 1 M hydrochloric acid at a concentration of 0.14% (v/v).

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Radiotracer synthesis [11C]Erlotinib was synthesized as described elsewhere.30 For i.v. injection into animals, [11C]erlotinib was formulated in 0.1 mM hydrochloric acid in physiological saline (0.9%, w/v). Molar activity at the time of injection was 60.6 ± 32.9 GBq/μmol and radiochemical purity was > 98%.

Animals Female FVB mice were obtained from Charles River (Sulzfeld, Germany). At the time of experiment, animals were 6-15 weeks old and weighed 23.1 ± 1.5 g. In total, 81 mice were used in the experiments. All animals were housed in type III IVC cages under controlled environmental conditions (21.8 ± 1.0°C, 40% to 70% humidity, 12-hour light/dark cycle) with free access to standard laboratory animal diet (ssniff R/M-H, ssniff Spezialdiäten GmbH, Soest, Germany) and water. An acclimatization period of at least 1 week was allowed before the animals were used in the experiments. The study was approved by the national authorities (Amt der Niederösterreichischen Landesregierung) and study procedures were in accordance with the European Communities Council Directive of September 22, 2010 (2010/63/EU). The animal experimental data reported in this study are in compliance with the ARRIVE (Animal Research: Reporting in Vivo Experiments) guidelines.

Experimental design An overview of examined animal groups and numbers is given in Table 1. Animals underwent 60-min dynamic [11C]erlotinib PET scans. At 30 min before start of the PET scan, animals received via a tail vein an i.v. bolus injection of either the respective ABCB1/ABCG2 inhibitor or vehicle solution over 1 min followed by continuous i.v. infusion

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of the ABCB1/ABCG2 inhibitor or vehicle solution until the end of the PET scan (total infusion time: 90 min) (for inhibitor doses see Table 1).

PET imaging Imaging experiments were performed under isoflurane anesthesia (1.5-2.5% in oxygen). Animals were warmed throughout the experiment and body temperature (37.5-38.5 °C) and respiratory rate (30-60 breaths/second) were constantly monitored. Mice were placed in a custom-made imaging chamber and both lateral tail veins were cannulated for i.v. administration. A microPET Focus220 scanner (Siemens Medical Solutions, Knoxville, TN, USA) was used for PET imaging. At 30 min before start of the PET scan, animals received via one of the catheters the respective inhibitor or vehicle solution (total volume: 100 µL) as an i.v. bolus over 1 min followed by a continuous infusion of inhibitor or vehicle solution until the end of the PET scan (Harvard Apparatus Syringe pump 11 Elite, total duration: 90 min, total volume: 150 µL). Dynamic emission scans (60 min) were started with the i.v. injection of [11C]erlotinib (29.9± 5.8 MBq in a volume of 100 µL, corresponding to 0.8 ± 0.5 nmol of unlabeled erlotinib) via the second tail vein catheter. List-mode data were acquired with a timing window of 6 ns and an energy window of 250-750 keV. At the end of the PET scan, a terminal blood sample was withdrawn from the retro-bulbar plexus and animals were sacrificed by cervical dislocation while still under deep anesthesia and whole brains were removed. Radioactivity in a blood aliquot and in whole brain was measured in a gamma counter (Wizard 1470, Perkin Elmer or Hidex Automatic Gamma Counter, Hidex). Blood was centrifuged to obtain plasma, which was stored at -80°C until analysis of inhibitor concentrations.

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PET data analysis The dynamic PET data were binned into 23 frames, which incrementally increased in time length. PET images were reconstructed using Fourier re-binning of the 3-dimensional sinograms followed by a 2-dimensional filtered back-projection with a ramp filter giving a voxel size of 0.4 × 0.4 × 0.796 mm³. Using AMIDE software,31 brain, liver, gall bladder, duodenum, intestine, left kidney, urinary bladder and right lung were manually outlined on the PET images (see Supporting Information, Figure S1) to derive time-activity curves expressed in units of standardized uptake value (SUV = (radioactivity per g/injected radioactivity) × body weight). In addition, the left ventricle of the heart was outlined to obtain an image-derived blood curve. It was assumed that the sum of radioactivity in the gallbladder, the duodenum and the intestine represented biliary radioactivity excreted from the liver. A graphical analysis method (integration plot)

32-34

was used to estimate the rate

constants for transfer of radioactivity from blood into brain (kuptake,brain, mL/min/g brain), liver (kuptake,liver, mL/min/g liver), kidney (kuptake,kidney, mL/min/g kidney) and lung (kuptake,lung, mL/min/g lung) using data measured from 0.3 min - 3.5 min (brain), 0.2 min -2.5 min (liver, kidney) and 0.8 min - 4.5 min (lung) after radiotracer injection and the following equation:

𝐶t,organ 𝐶t,blood

= 𝑘uptake,organ ×

𝐴𝑈𝐶0 ― t,blood 𝐶t,blood

+ 𝑉E

where Ct,organ is the radioactivity concentration in brain, liver, kidney or lung at time t and Ct,blood is the radioactivity concentration in the left ventricle of the heart at time t. AUC0-t,blood represents the area under the time-activity curve in the left ventricle of the heart from time 0 to time t. Kuptake,organ can be obtained by performing linear regression analysis of a plot of Ct,organ/Ct,blood versus AUC0-t,blood/Ct,blood and calculating the slope of the regression line (see Supporting Information, Figure S2). VE is the y-intercept of the integration plot. 10


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Moreover, the rate constants for transfer of radioactivity from kidney into urine (kurine, 1/min) and from liver into bile (kbile, 1/min) were determined from 12.5 min - 45 min after radiotracer injection using the following equation:

𝐶t,organ = 𝑘fluid × 𝐴𝑈𝐶0 ― t,organ + 𝑉E

where Ct,organ is the radioactivity concentration in urinary bladder or in the intestine (including gall bladder and duodenum) at time t. AUC0-t,organ represents the area under the time-activity curve in the kidney or liver from time 0 to time t. Kfluid (kurine or kbile) can be obtained by performing linear regression analysis of a plot of Ct,organ versus AUC0-t,organ and calculating the slope of the regression line (see Supporting Information, Figure S2). VE is the y-intercept of the integration plot. Total distribution volume (VT, mL/cm3) in brain, liver, kidney and lung was estimated by using Logan graphical analysis over the interval of 6.25 min - 45 min after radiotracer injection.35 VT corresponds to the organ-to-blood ratio of radioactivity at steady state.

Determination of drug concentrations in plasma Erlotinib concentrations in plasma were determined as described elsewhere.36 The concentration of imatinib, lapatinib and tariquidar was determined by high-performance liquid chromatography (HPLC) using a Dionex UltiMate 3000 system (Thermo Fisher Scientific, Inc., Waltham, MA) with ultraviolet detection at 266 nm for imatinib, at 331 nm for lapatinib and at 240 nm for tariquidar. Frozen samples were thawed at room temperature. After the addition of 150 µL ice-cold methanol to 50 µL of plasma, the samples were centrifuged (13000 g for 5 min at 4°C) and 100 µL of the clear supernatants were injected onto a Hypersil BDS-C18 column (5 µm, 250 × 4.6 mm I.D., Thermo Fisher Scientific, Inc.,

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Waltham, MA), preceded by a Hypersil BDS-C18 guard column (5 µm, 10 × 4.6 mm I.D.) at a flow rate of 1.0 mL/min. The column oven was set at 40°C. The mobile phase consisted of a continuous gradient mixed from ammonium acetate buffer (10 mM, pH = 5.0) (mobile phase A) and acetonitrile (mobile phase B). Mobile phase B linearly increased from 25% (0 min) to 50% at 10 min for imatinib and from 30% (0 min) to 70% at 10 min for lapatinib and tariquidar. Subsequently, mobile phase B was further increased to 90% at 11 min, and kept constant at 90% until 15 min. The percentage of acetonitril was then decreased within 1 min to 25% and 30%, respectively, to equilibrate the column for 6 min before application of the next sample. Linear calibration curves were performed from the peak areas of imatinib, lapatinib and tariquidar by spiking drug-free mouse plasma with standard solutions of imatinib, lapatinib and tariquidar to obtain a concentration range of 0.1 to 50 µg/mL (calculated (average correlation coefficients > 0.998). For these methods, the limit of quantification for all three drugs was determined to be 100 ng/mL in mouse plasma (coefficients of accuracy and precision were < 9.1%).

Analysis of radiolabeled metabolites FVB mice, pre-treated either with tariquidar or lapatinib or the respective vehicle solutions as in the PET experiments (see Table 1), were injected under isoflurane anesthesia with [11C]erlotinib (30.9 ± 8.3 MBq in a volume of 100 µL, corresponding to 0.3 ± 0.1 nmol of unlabeled erlotinib) as an i.v. bolus over 1 min. At 25 min after [11C]erlotinib injection a terminal blood sample was withdrawn under isoflurane anesthesia from the retro-bulbar plexus and the animals were sacrificed by cervical dislocation. Whole brain, liver, kidneys and gall bladder were sampled and urine was collected. Blood was centrifuged to obtain plasma. Organs and fluids were counted for radioactivity in a gamma counter and radiolabeled

metabolites

of

[11C]erlotinib

12

were

assessed


with

radio-thin

layer

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chromatography (radio-TLC) as described in detail elsewhere.14 The 25 min time point was chosen for metabolite analysis because it represented a time point in the middle of the PET scan at which radioactivity counts were still high enough to allow for quantification. . Statistical analysis All values are given as mean ± standard deviation (SD). Statistical testing was performed using Prism 8.0 software (GraphPad Software, La Jolla, CA, USA). Differences in outcome parameters between two groups were tested with a 2-sided unpaired t-test and between multiple groups with 1-way ANOVA with Dunnett’s multiple comparisons test. To assess correlations, the Pearson correlation coefficient r was calculated. The level of statistical significance was set to a P value of less than 0.05.

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Results The inhibitors tested in the present study were administered as a short i.v. bolus injection at 30 min before start of the PET scan followed by continuous i.v. infusion for the duration of the PET scan. Table 1 gives an overview of the tested inhibitors and the administered doses. As outcome parameters for brain distribution of [11C]erlotinib we determined VT,brain and kuptake,brain (Figure 1A, Supporting Information, Figure S2). For determination of both parameters, the time-activity curve in arterial blood needs to be known. As arterial blood sampling is difficult to perform in mice, we generated an image-derived blood curve by placing a region of interest into the left ventricle of the heart. PET-derived blood radioactivity measurements showed a good correlation with radioactivity measured with a gamma counter in a venous blood sample collected at the end of the PET scan, which validated our approach to generate an image-derived blood curve (Figure 2A). Also brain radioactivity concentrations measured with PET showed a good correlation with the respective gamma counter values (r = 0.778, P < 0.0001, not shown). In the Supporting Information, Figure S3 and in Figure 3 time-activity curves in blood (image-derived) and in brain are shown for all vehicle-and inhibitor-treated groups. VT,brain and kuptake,brain values in different inhibitor-treated animals and their respective vehicle-treated controls are shown in Figure 4. Five out of eight tested drugs increased VT,brain values as compared with controls (tariquidar: +69%, erlotinib: +19% and +23% for the 21.5 mg/kg and the 43 mg/kg dose, respectively, imatinib: +22%, lapatinib: +25% and cyclosporine A: +49%) (Figure 4A). For comparison, in Abcb1a/b(-/)Abcg2(-/-)

mice VT,brain was +149% higher than in wild-type mice.14 For most groups, in which

VT,brain values were increased, kuptake,brain values were also significantly increased (Figure 4B). Radio-TLC analysis revealed that the majority of radioactivity in the brain (> 80%) was composed of unmetabolized [11C]erlotinib without any significant differences between

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vehicle-treated mice and mice treated with selected inhibitors (tariquidar and lapatinib) (Table 2). In some of the inhibitor-treated groups total blood radioactivity concentrations measured at the end of the PET scan in a gamma counter were significantly higher as compared with the respective vehicle groups (erlotinib: +103% and +113% for the 21.5 mg/kg and the 43 mg/kg dose, respectively, imatinib: +125% and cyclosporine A: +101%) (Figure 2B, Supporting Information, Figure S3). To elucidate the causes of increased blood radioactivity concentrations following inhibitor treatment, we assessed radioactivity distribution to the clearance organs liver and kidney as well as the rate constants for biliary and urinary secretion of radioactivity (kbile and kurine) (Figure 1B and C, Supporting Information, Figure S2). In the Supporting Information, Figure S4 and Figure S5 time-activity curves in liver and intestine are shown for all groups. Figure 5 gives an overview of VT,liver, kuptake,liver and kbile values in different animal groups. VT,liver values were significantly decreased after erlotinib treatment (-18% and -29% for the 21.5 mg/kg and the 43 mg/kg dose, respectively) (Figure 5A). Kuptake,liver values were similar across all groups with the exception of pazopanibtreated animals, in which kuptake,liver was slightly but significantly decreased (-20%) (Figure 5B). Two inhibitors caused a significant decrease in kbile, i.e. erlotinib (-74% and -87% for the 21.5 mg/kg and the 43 mg/kg dose, respectively) and imatinib (-64%) (Figure 5C). We also studied distribution of radioactivity to the kidney and its urinary secretion (Figure 1C). In the Supporting Information, Figure S6 and Figure S7 time-activity curves in kidney and urinary bladder are shown for all groups. Figure 6 gives an overview of VT,kidney, kuptake,kidney and kurine values in different groups. Several inhibitors caused significant decreases in VT,kidney and/or kuptake,kidney values (osimertinib, tariquidar, erlotinib, nilotinib and imatinib). Conversely, cyclosporine A increased VT,kidney by +103% (Figure 6A and Figure 6B).

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Tariquidar and nilotinib caused pronounced increases in kurine values by +514% and +367%, respectively (Figure 6C). A similar trend for increased kurine values was also observed for lapatinib, but statistical significance was not reached. We finally studied distribution of radioactivity to the lungs as one pharmacological target tissue of erlotinib (Figure 1D, Figure 7, Supporting Information, Figure S8). VT,lung values were similar across all vehicle- and inhibitor-treated groups (Figure 7A). Kuptake,lung values were quite variable but not significantly different among groups, except for a significant decrease in tariquidar-treated animals by -60% (Figure 7B). We used radio-TLC to assess the percentage of radiolabeled metabolites of [11C]erlotinib in different organs and fluids of tariquidar- and lapatinib-treated mice and the respective vehicle controls (Table 2). The majority of radioactivity in plasma, brain, liver and kidney was composed of unchanged [11C]erlotinib, while the majority of radioactivity in bile and urine was composed of polar radiolabeled metabolites. Tariquidar treatment led to significant increases in the percentage of radiolabeled metabolites (corresponding to a decrease in the percentage of unchanged parent) in plasma, kidney and liver, while lapatinib treatment only led to a significant increase in the percentage of radiolabeled metabolites in plasma (Table 2). Table 3 summarizes inhibitor concentrations in mouse plasma and compares these values with clinical plasma concentrations. Plasma concentrations of erlotinib, imatinib and lapatinib were higher than clinically achievable concentrations, whereas for tariquidar they were in similar range as in humans.

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Discussion We assessed the inhibitory effect of seven marketed drugs and one non-marketed drug (tariquidar) on ABCB1 and ABCG2 at the mouse BBB by measuring brain uptake of the dual ABCB1/ABCG2 substrate [11C]erlotinib with PET. Using the PET approach we employed a microdose of [11C]erlotinib (< 1 µg) and not a clinically relevant dose. Even though previous studies had shown that [11C]erlotinib displayed non-linear pharmacokinetics,14,

15, 29

non-

linearity in brain distribution of erlotinib was observed at considerably higher than clinically used doses. It can therefore be expected that brain distribution of the microdose predicted brain distribution of a therapeutic dose reasonably well. Erlotinib was chosen as a model ABCB1/ABCG2 substrate, which was assumed to be representative of the behavior of other TKIs which are subject to efflux transport by ABCB1 and ABCG2 at the BBB. Erlotinib is approved for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) harboring activating mutations in the EGFR gene. Approximately 25-40% of these patients develop brain metastases, for which currently available treatment approaches are scarce. Erlotinib has limited efficacy to treat NSCLC brain metastases due to its poor penetration of the BBB. The third-generation EGFR-targeting TKI osimertinib, which shows better BBB permeability than erlotinib, has been proposed for more effective treatment of NSCLC brain metastases.37,

38

Nevertheless, the availability of a clinically

applicable ABCB1/ABCG2 inhibitor which can enhance brain delivery of erlotinib would be of great interest to treat NSCLC brain metastases as well as other brain tumors with EGFR overexpression (e.g. glioblastoma).6 In a previous study, we have observed that the ABCB1 inhibitory effect of the experimental inhibitor tariquidar was quickly reversible requiring this compound to be administered as a continuous i.v. infusion to achieve effective and sustained ABCB1 inhibition at the BBB.39 Accordingly, we administered in the present study all tested drugs to

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mice as a continuous i.v. infusion for the duration of the PET scan. All compounds were administered at their maximum solubility. As solubility differed among drugs, we had to employ three different vehicle solutions, for which appropriate control groups were included. As outcome parameters of [11C]erlotinib brain distribution we determined VT,brain and kuptake,brain, which were shown in previous studies to reflect ABCB1/ABCG2 transport activity at the BBB.14,

15

Both parameters were estimated with graphical analysis approaches

employing an image-derived arterial blood curve. To assess maximum possible brain uptake of [11C]erlotinib in absence of ABCB1 and ABCG2, we considered previously published PET data in Abcb1a/b(-/-)Abcg2(-/-) mice.14 These mice showed +149% and +358% higher VT,brain and kuptake,brain values, respectively, as compared with wild-type mice. On the other hand, Abcb1a/b(-/-) mice and Abcg2(-/-) mice showed relative to wild-type mice only +30% and +16% increases in VT,brain values and +71% and +62% increases in kuptake,brain values, respectively, which was in line with the typical behavior of a dual ABCB1/ABCG2 substrate.14 In our previous work, we had shown in FVB mice that i.v. bolus co-injection of unlabeled erlotinib (10 mg/kg) with [11C]erlotinib led to a marked increase in kuptake,brain, while VT,brain was unchanged.14 This lack of an effect of erlotinib co-injection on VT,brain could most likely be attributed to the reversibility of the ABCB1/ABCG2 inhibitory effect of erlotinib. In the present work, continuous i.v. infusion of erlotinib at a total dose of 21.5 mg/kg led to a +19% increase in VT,brain. Erlotinib plasma concentrations at the time of the PET scan (18.9 ± 3.5 μmol/L) were comparable to erlotinib plasma concentrations in a recently performed study in non-human primates (16 µmol/L), in which erlotinib was administered in a comparable continuous i.v. infusion scheme as in the present study.15 In the non-human primate study, VT,brain of [11C]erlotinib was +73% higher as compared with PET scans without erlotinib infusion. This difference in the magnitude of the effect of erlotinib

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infusion on [11C]erlotinib brain distribution can most likely be attributed to erlotinib being a more potent inhibitor of ABCG2 than of ABCB1

28

in combination with species differences

in the relative transporter abundance at the non-human primate and mouse BBB. In mice, ABCB1A is the predominant ABC transporter at the BBB with an ABCB1A/ABCG2 ratio of approximately 3.40 In non-human primates, on the other hand, ABCG2 appears to be, similar to humans, the predominant ABC transporter with an ABCB1/ABCG2 ratio of approximately 0.3 reported in cynomolgus monkeys.41 Hence, a relatively greater inhibition of ABCG2 than of ABCB1 is expected to lead to greater increases in brain distribution of dual ABCB1/ABCG2 substrate drugs in non-human primates and humans than in mice. In accordance with this, in humans VT,brain increase of [11C]erlotinib after oral intake of 650 mg of unlabeled erlotinib (+23%) was comparable to the VT,brain increase in mice, although erlotinib plasma concentrations were substantially lower in humans (6.7 ± 2.0 µmol/L) than in mice.29 We also studied a second group of mice, which was infused with 43 mg/kg of erlotinib. In these animals, erlotinib plasma concentrations were approximately two-fold higher (38.0 ± 12.5 μmol/L) than in the first group. However, the increase in VT,brain of [11C]erlotinib was only slightly higher (+23%) than in animals of the first group. We also studied the experimental, non-marketed ABCB1 inhibitor tariquidar.42 In contrast to previously published data in humans, where continuous tariquidar infusion had no effect on [11C]erlotinib brain distribution,29 we observed a VT,brain increase of +69% in mice at comparable plasma concentrations as in humans (Table 3). Tariquidar is a more potent inhibitor of ABCB1 than of ABCG2 and preferentially inhibits ABCB1 at clinically achievable plasma concentrations.43 Hence, this difference in the effect of tariquidar on [11C]erlotinib brain distribution between mice and humans may be related to the relatively higher abundance of ABCB1 than that of ABCG2 at the mouse BBB as compared with the human BBB.44

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Next to tariquidar, we assessed the immunomodulatory drug cyclosporine A, which has been employed in previous studies as an inhibitor of ABCB1 at the BBB

45

and which

was also shown to inhibit ABCG2.46 Cyclosporine A led to a +49% increase in VT,brain as compared with vehicle-treated animals. However, the employed cyclosporine A dose (50 mg/kg) was several-fold higher than a clinically feasible i.v. dose (5 mg/kg), so that it is questionable if cyclosporine A can increase brain uptake of erlotinib in humans. We assessed a panel of marketed TKIs with known ABCB1/ABCG2 inhibitory properties based on published data (osimertinib, nilotinib, imatinib, lapatinib and pazopanib).20-23 Among these compounds the highest increases in VT,brain and kuptake,brain values were obtained for imatinib and lapatinib. However, whereas lapatinib plasma concentrations were several-fold higher than clinically achievable plasma concentrations, imatinib plasma concentrations were only two-fold higher than in humans (Table 3). This suggests that imatinib may have some potential to be repurposed as an ABCB1/ABCG2 inhibitor to enhance brain distribution of dual ABCB1/ABCG2 substrates in humans; however, these findings need to be further confirmed with clinically relevant doses of the dual ABCB1/ABCG2 substrates. Interestingly, previous studies have shown that imatinib restores BBB integrity and reduces infarct size, hemorrhagic transformation and cerebral edema in stroke models treated with tissue plasminogen activator.47 High-dose imatinib has been suggested as potential treatment aid to acute ischemic stroke.48 In this context, the ability of imatinib to inhibit ABCB1/ABCG2 at the BBB and enhance brain delivery of concomitantly administered dual ABCB1/ABCG2 substrates needs to be considered. ABCB1 and ABCG2 are not only expressed at the BBB but also in clearance organs (liver and kidney) and therefore their inhibition may affect the peripheral pharmacokinetics of transporter substrates. Changes in peripheral pharmacokinetics may lead to an increase in systemic side effects of the employed drugs. We observed that some of the tested inhibitors

20


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(erlotinib, imatinib and cyclosporine A) led to significant increases in blood concentrations of total radioactivity measured at the end of the PET scan (Figure 2B). In order to elucidate the mechanism by which the tested inhibitors increased total blood radioactivity concentrations we assessed the rate constants for hepatobiliary excretion of radioactivity, which is the main excretion route for erlotinib.49 [11C]Erlotinib has been identified as a substrate of the basolateral hepatocyte uptake transporter organic anion-transporting polypeptide 2B1 (SLCO2B1), which may promote its uptake from blood into liver.36, 50 In the present study, kuptake,liver values were largely unchanged among different inhibitor-treated groups, which argued against an inhibition of hepatocyte uptake transporters as an explanation for the increased blood radioactivity concentrations. In contrast, some of the tested inhibitors led to marked decreases in kbile, most likely due to inhibition of canalicular hepatocyte efflux transporters, such as ABCG2, which mediate biliary secretion of [11C]erlotinib and possibly its radiolabeled metabolites.34 It is noteworthy that in some of the inhibitor-treated groups an increase in the rate constant for urinary excretion of radioactivity (kurine) was observed, most notably for tariquidar, nilotinib and lapatinib (Figure 6C). In a previous study 14 and in the present study (Table 2) we have shown that the majority of radioactivity excreted into urine is in the form of unidentified polar radiolabeled metabolites of [11C]erlotinib. It appears possible that decreased biliary secretion of [11C]erlotinib increased the availability of [11C]erlotinib to metabolic enzymes in hepatocytes leading to an increased formation of radiolabeled metabolites. An increased amount of radiolabeled metabolites could in turn lead to an increase in kurine values, provided that urinary secretion of the radiolabeled metabolites is promoted by transporters in the brush border membrane of kidney proximal tubule cells, which do not transport the parent compound. We assessed radiolabeled metabolites of [11C]erlotinib in separate groups of tariquidar-and lapatinib-treated animals and indeed

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observed significant increases in the percentage of radiolabeled metabolites in plasma, kidney and liver for tariquidar and in plasma for lapatinib (Table 2), which supported our assumption of enhanced metabolism as an explanation for increased urinary excretion of radioactivity. Such an interplay between hepatic transporters and enzymes, most notably between ABCB1 and CYP3A4, in drug disposition has also been observed before for other drugs.51,

52

Enhanced metabolism in response to canalicular efflux transporter inhibition in hepatocytes may counteract the effect of transporter inhibition at the BBB, as it may lead to a decrease in systemic concentrations of intact parent drug available for crossing the BBB. It should be noted, however, that the pharmacokinetic processes (e.g. transport, metabolism) playing an important role in the disposition of the PET microdose may be partly saturated for the therapeutic dose, so that not all of our findings may extrapolate to a therapeutic dose of the drug. Our study illustrates the difficulty to achieve complete ABCB1/ABCG2 inhibition at the BBB. Potent in vitro ABCB1/ABCG2 inhibitors may not necessarily translate into effective inhibitors at the BBB in vivo. In this framework, PET imaging using radiolabeled analogues of drugs is an appealing method to evaluate strategies to enhance the brain delivery of ABC transporter substrates in vivo.53 The partial transporter inhibition observed with the tested inhibitors suggests that a crucial factor for ABCB1/ABCG2 inhibition protocols at the BBB are the concentration levels to be achieved in vivo. The choice of a marketed drug to be repurposed as an ABCB1/ABCG2 inhibitor should therefore consider both the in vitro transporter inhibition potencies and the possibility to safely reach high enough plasma concentrations. Furthermore, our data show that administration of transporter inhibitors for improvement of drug brain delivery can lead to transporter-mediated drug-drug interactions in clearance organs. This may have effects on the systemic and organ pharmacokinetics of the test drugs, lead to additional toxicities and therefore needs to be considered when employing

22


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drug combinations.17 Moreover, our data highlight the peripheral drug interaction potential of combination therapies of molecularly targeted drugs. Small-animal PET is a powerful tool to elucidate transporter-mediated drug-drug interactions on a whole-body level.54 However, a limitation of PET is that total radioactivity concentrations are measured (including parent drug and radiolabeled metabolites), so that changes in drug metabolism cannot be detected. In conclusion, we tested several different marketed drugs for their ability to increase brain uptake of [11C]erlotinib assessed with PET imaging in mice. Five out of eight tested drugs led to significant increases in brain uptake of [11C]erlotinib. However, these increases were considerably lower than those in transgenic mice lacking ABCB1 and ABCG2, suggesting that only partial ABCB1/ABCG2 inhibition could be achieved at the mouse BBB. In addition, the plasma concentrations of the tested drugs were higher than clinically achievable plasma concentrations, which raises questions with regards to the clinical translatability of our results. Some of the tested drugs also led to increases in blood radioactivity concentrations, most likely due to inhibition of hepatobiliary excretion. Our data highlight the challenges in employing transporter inhibitors to increase brain delivery of transporter substrates.

Acknowledgments This work was supported by the Lower Austria Corporation for Research and Education (NFB) [grant LS15-003, to O. Langer]. The authors would like to thank Mathilde Löbsch (AIT) for assistance in the animal experiments.

23



Supporting Information Time-activity curves in blood, liver, intestine, kidney, urinary bladder and lung for all vehicle-and inhibitor-treated groups. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure Legends Figure 1. Outcome parameters from graphical analysis approaches. VT,brain, VT,liver, VT,kidney, VT,lung (mL/cm3), total distribution volume in brain, liver, kidney and lung, respectively, determined with Logan graphical analysis. kuptake,brain, kuptake,liver, kuptake,kidney, kuptake,lung (mL/min/g tissue), rate constants for transfer of radioactivity from blood into brain, liver, kidney and lung, respectively, determined with integration plot analysis. kbile, kurine (1/min), rate constants for biliary and urinary secretion of radioactivity, respectively, determined with integration plot analysis.

Figure 2. Correlation of radioactivity concentration (SUV) in the left ventricle of the heart measured in the last PET time frame (50-60 min) with radioactivity concentration (SUV) in venous blood collected at the end of the PET scan measured in a gamma counter. Broken line represents linear regression fit (r = Pearson correlation coefficient) (A). Total radioactivity concentration (SUV) in venous blood collected at the end of the PET scan, measured in a gamma counter in different animal groups (B). *, P < 0.05, ** P < 0.01, *** P < 0.001, 1way ANOVA with Dunnett’s multiple comparisons test.

Figure 3. Time-activity curves (mean + SD) in whole brain for all vehicle-and inhibitortreated groups. Radioactivity is expressed as standardized uptake value (SUV).

Figure 4. Total distribution volume of radioactivity in the brain (VT,brain, mL/cm3) (A) and rate constant for transfer of radioactivity from blood into brain (kuptake,brain, mL/min/g brain) (B) for different animal groups. For comparison, previously acquired data in Abcb1a/b(-/)Abcg2(-/-)

mice

14

are also shown (mean value indicated by dotted line). *, P < 0.05, ** P
99

39

9 (p.o.)

9.1 ± 2.2

95

29

Erlotinib 2

38.0 ± 12.5 c

Imatinib

25.2 ± 5.0 c

9 (p.o.)

13.7

99

55, 56

Lapatinib

22.5 ± 4.8 b

21 (p.o.)

4.5

>99

57

Stated is the mean ± SD concentration (µmol/L, n = 4 for all groups except 43 mg/kg erlotinib with n = 2) of the respective inhibitor determined with HPLC in mouse plasma, dose, maximum plasma concentrations and percentage plasma protein binding reported in the literature for healthy subjects or patients. p.o., per oral, i.v., intravenous a

based on a body weight of 70 kg

b

determined at 55 min after start of the infusion (corresponding to 25 min after start of PET

scan) c

determined at 90 min after start of the infusion (corresponding to end of PET scan)

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Figure 1

39



Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 1 338x190mm (96 x 96 DPI)


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Figure 2 82x115mm (300 x 300 DPI)



Figure 3 177x131mm (300 x 300 DPI)


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Figure 4 82x118mm (300 x 300 DPI)



Figure 5 82x172mm (300 x 300 DPI)


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Figure 6 82x173mm (300 x 300 DPI)



Figure 7 82x120mm (300 x 300 DPI)


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Graphical abstract 266x107mm (96 x 96 DPI)


Inhibition of ABCB1 and ABCG2 at the mouse blood-brain barrier with

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